Yokenella regensburgei urinary tract infection in an immunocompetent patient: a case report

Yokenella regensburgei , belonging to the order Enterobacterales , is a rare and emerging human pathogen reported to cause both superficial and invasive infections. The 13 case reports in the literature worldwide highlight blood, bone and wound infections. To our knowledge this is the first case description of Y. regensburgei causing a urinary tract infection in a 69-year-old immunocompetent patient which was isolated in two separate specimens and identified using matrix-assisted laser desorption ionization time-of-flight MS. It was found to be susceptible to most antimicrobials but resistant to penicillin, amoxicillin-clavulanate, cefoxitin and colistin. Inducible chromosomal ampC resistance was demonstrated on disc approximation testing, and blaYOC-1 class C beta-lactamase, beta lactamase superfamily and MBL fold metallo-hydrolase genes were found on whole genome sequencing.


InTRODuCTIOn
Yokenella regensburgei, a Gram-negative bacillus belonging to the order Enterobacterales, is the only recognized species of the genus Yokenella [1].It has been isolated from the intestine of insects, reptiles and well water from the environment.Y. regensburgei has been reported to be a rare pathogen causing infection mainly in immunocompromised patients.It phenotypically resembles Hafnia alvei and has been isolated worldwide from blood, wounds, bone and joint aspirates in humans [1,2].Clinical presentation of a patient with urinary tract infection (UTI) associated with this organism has not been described in the literature although a solitary report mentions isolation from urine [3].Thus, we present and discuss the first case identified at our centre of Y. regensbergei causing a UTI.

Case details
A 69-year-old retired man, with benign prostatic hypertrophy (BPH), had undergone a transurethral resection of the prostate 5 years previously.A year later he developed poor urine flow, straining to void and incomplete voiding.He had developed near urinary retention and UTI multiple times in the previous 2 years.He was diagnosed to have a urethral stricture and had undergone urethral dilatation elsewhere a year previously.In the 2 weeks prior to presentation at our centre, he had complained of increased frequency, burning micturition and dribbling.He did not have fever.On investigation, he was found to have a tight bulbar stricture on cystoscopy and micturating cystourethrogram (MCU) with a high post-void residue.Ultrasonography showed an enlarged prostate with no hydronephrosis.His renal functions were altered with an estimated glomerular filtration rate of 42 ml min −1 /1.73 m 2 and serum creatinine was elevated at 1.76 mg%.
He had no other no comorbidities.His fasting glucose was 116 mg dl −1 and 2 h post-prandially this was 154 mg dl −1 .His haemoglobin was 14.8 g% and he was not on any medication.He did not give any history of smoking or alcohol intake.Investigations did not reveal any blood-borne virus.urine analysis, culture and susceptibility Urine analysis detected elevated white blood cells (WBCs), leukocytes and nitrates (urine routine analysis: WBC: 31 per high-power field, bacteria: 2+, leukocytes: 2+, nitrates: 1+).A concomitant clean mid-stream semi-quantitative urine culture was performed with 10 µl of the specimen on sterile and quality-passed 7 % sheep blood agar and MacConkey agar media, prepared in the Department of Clinical Microbiology.Significant growth indicated by >100 000 c.f.u.ml −1 Y. regensbergei was isolated in culture at 37 °C under ambient air, and the Gram-stained smears on the specimen were consistent with an inflammatory response.Three days later a suprapubic urine aspirate (SPA) was sent for culture to confirm the findings of mid-stream collection.The same culture findings were obtained on the SPA specimen.

Detection
Preliminary screening of the isolate revealed a glucose-and mannitol-fermenting, motile organism, which was lactose-and sucrose non-fermenting, and indole was not produced.Citrate was utilized after 48 h of incubation [MMTPC: ++−/(+)−+].Final identification was determined by complete biochemical characterization and by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS (bioMérieux; Version 3.2) with a 99.9 % high-confidence identification.
Multiplex PCRs to detect plasmid-mediated extended-spectrum beta-lactamase (ESBL) (TEM, SHV, VEB, PER, GES, SPM) and carbapenem resistance genes (KPC, NDM, VIM, IMP, OXA-48like) were negative.Multiplex PCR for the genes CMY/MOX, CIT, DHA, ACC, ACT/MIR and FOX, as described by Pérez-Pérez FJ and Hanson 2002 [5], to determine carriage of chromosomally mediated ampC genes, was also negative.Phenotypic detection of a chromosomally encoded ampC gene in the presence of a potent inducer of ampC (cefoxtin) revealed a positive result as indicated by a flattening of the zone of inhibition around cefotaxime 30 µg when placed close to the inducer 30 µg cefoxitin (disc approximation test) (Fig. 1).Since PCR did not detect the presence of ampC genes, whole genome sequencing was performed using the Ilumina platform and raw reads were assembled using skesa and annotated with PGAP and eggNOG.This revealed blaYOC-1 class C beta-lactamases, beta-lactamase superfamily and MBL fold metallo-hydrolase genes, leading to the beta-lactam resistance as observed phenotypically, while the arnD, arnT and pmrD genes were identified by eggNOG contributing to colistin resistance (BioProject accession number PRJNA926362).

Outcome
Following the investigations, the patient underwent a suprapubic catheter placement and subsequently was treated with cefaperazone-sulbactam for 5 days (1.5 g intravenously).His symptoms resolved and he was planned for a substitution urethroplasty.

DISCuSSIOn
Y. regensburgei was initially identified as NIH biogroup 9 by the National Institute of Japan and as enteric group 45 by the Centers for Disease Control and Prevention (CDC).In 1984 it was named as Y. regensburgei by Kosako et al., while the CDC named it Koserella trabulsii replacing enteric group 45.Later, both these organisms were found to represent the same organism and the name Y. regensburgei had priority over K. trabulsii based on the Bacteriological Code and the CDC approved Y. regensburgei in 1991 [1,4].It has been usually isolated from insect intestine, reptiles, raw salads and well water, while from humans it has been isolated worldwide, from superficial and invasive site specimens.
Y. regensburgei as a clinical pathogen is not well described because of the difficulty in identifying it in low-resource laboratories.It is a nondescript short Gram-negative bacillus and grows as non-haemolytic, non-mucoid grey colonies on sheep blood agar (7 %) and is non-lactose-fermenting on MacConkey agar (Fig. 2).
A wide range of biochemicals or automated identification systems are required to identify the organism as it closely resembles and can be misidentified as Hafnia alvei, Enterobacter spp.and Serratia spp., due to its biochemical similarity.

2021) and osteoarticular infection (Guilarde, 2021
).There is no report describing UTIs associated with the bacterium, although Aziz [3] reports its isolation from urine Table 1.Identification of rare pathogens such as this has been made possible with the help of automated machines such as bioMérieux's Vitek-2 and MALDI-TOF MS, BD Phoenix or a wide array of biochemicals.Of the 13 available reports, nine investigators used newer automated or molecular identification methods, all these reports coming in the last decade (Table 1).The unique protein pattern of rarer pathogens aids in high-confidence identifications.
For In our tertiary care centre and high-capacity laboratory, which receives approximately 50 000 urine specimens annually with all organism identifications made with a wide range of biochemicals, VITEK-2 or MALDI-TOF MS or organism-specific PCR, this is the first isolate of Yokenella identified, highlighting its rarity.It was isolated in two specimens sent 3 days apart, the second of which was a direct suprapubic aspirate, confirming its presence and pathogenicity in the urinary tract.Further, of the 17 WGS deposits of this organism in the genome database only four were isolated from humans and none have been reported from UTIs, also highlighting its rarity despiteits world-wide pathogenic potential.Finally, our patient, a known case of urethral stricture with recent symptoms of cystitis, was treated with cefaperazone-sulbactam intravenously for 10 days, which is one of the standard treatment options in the management of complicated urology cases in our centre.His symptoms resolved and he was planned for a substitution urethroplasty, which proceeded uneventfully.

COnCluSIOn
Y. regensbergei is a rare pathogen that can invade the urinary tract irrespective of the host's immune status.It can be identified by both complete conventional biochemicals and automated systems.The key to its identification is its resistance to penicillins, cefoxitin, amoxicillin-clavulanate and colistin.Genotypically, it carries the ampC gene chromosomally, and thus third-generation cephalosporins are best avoided in the management of these infections.
To our knowledge, this is the first case description of Y. regensbergei causing a UTI.
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your submissions to our titles support the community -ensuring that we continue to provide events, grants and professional development for microbiologists at all career stages.No.
The first specimen, at first OPD visit, was the voided -mid-stream collection.
A pure (single organism) significant growth (>100,000 colonies/ml of urine) was isolated in culture of Yokenella regensbergei.
To confirm, a suprapubic aspirate was obtained 3 days later, which grew the same organism.
2 Line stating "he had no other significant history" misleading The line has been modified.He had no other no comorbidities.His fasting glucose was 116 mg/dl and 2 hours post-prandial was154mg/dl.His hemoglobin was 14.8gm% and he was not on any medications.

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Reviewer 1
Did the patient have a previous history of UTI? Yes.

4.
Did the infection appear before or after suprapubic aspiration?
Before the aspiration 5. Do you think that the suprapubic has a role for causing the infection? No.
The first specimen, at first OPD visit, was the voided -midstream collection.
A pure (single organism) significant growth (>100,000 colonies/ml of urine) was isolated in culture of Yokenella regensbergei.
To confirm, a suprapubic aspirate was obtained 3 days later, which grew the same organism.

6.
Figure 1 for antibiotics, ament (IMPROVE) title for inhibitory action of antibiotics Please add the funding information state in the main manuscript to the online submission system.
The funding information as stated on lines 182-184 will be added to the online submission system.
We have tried our best to address the questions/suggestions.We hope that you find them answered satisfactorily.

Fig. 1 .
Fig. 1.Phenotypic detection of the inducible chromosomal ampC gene via a disc approximation test.

Table 1 .
Yokenella regensburgei previously reported in the published literature [6][7][8]al identification purposes, Yokenella is motile, ferments the carbohydrates mannitol, glucose, arabinose, rhamnose, maltose, xylose and trehalose but not lactose, sucrose, dulcitol, adonitol, inositol or sorbitol.It is methyl red test-positive.It produces gas on fermentation and utilizes citrate slowly, which can be detected after 48 h.It also decarboxylates the amino acids lysine and ornithine.As a strong fermenter, Yokenella can be differentiated from H. alvei by the methyl red test, citrate utilization, and inositol and sorbitol fermentation, from Enterobacter spp.by the methyl red test and lysine decarboxylation, from Serratia spp.by the methyl red test, malonate, arabinose, gelatin liquefaction and DNAase test[6][7][8], and in susceptible strains by their characteristic antimicrobial resistance.A study by Stock et al. on 10 strains of Y. resenburgei found that, genotypically, all members of the species had the ampC gene and strongly expressed inducible β-lactamses.Their study also reported that Yokenella species were resistant or had intermediate susceptibility to penicillin G, oxacillin, amoxicillin, amoxicillin-clavulanate, cefalcor, cefoxitin, fusidic acid, linezolid, glycopeptides, rifampicin, fusidic acid, streptogramins and lincosamides but were sensitive to several β-lactams, aminoglycosides, [6,7]amphenicol, cotrimoxazole, fosfomycin and tetracyclines[6,7].Our isolate had a similar resistance profile, with a positive disc approximation test detecting inducible ampC gene carriage and whole genome sequencing (WGS) identified blaYOC-1 class C beta-lactamases.

recommendation and comments https
The work presented is clear and the arguments well formed.This study would be a valuable contribution to the existing literature.The reviewers have highlighted minor concerns with the work presented.Please ensure that you address their comments.The reviewers found your case report interesting and valuable and have suggested some minor amendments to improve the manuscript.Please address all reviewer comments in a revised manuscript.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.The case description was thorough with details especially in identification of the organism and antimicrobial susceptibility testing.The case was straight forward, but as it was a rare pathogen, it was worth sharing among colleagues, especially being the first documented case of causing UTI.Literature review was meticulous and summaries of each case was very useful.Discussion was detailed and compared with previous literatures.Overall, it was a great case report.As a minor amendment, I would like the author to include the dosage and detailed route of cefaperazone-sulbactam given and mention other blood investigations results if performed such as CRP/ESR or procalcitonin and full blood count: these to correlate the positive microbiology result with patient's systemic response and parameters.://doi.org/10.1099/acmi.0.000571.v1.5 © 2023 Abou El-Khier N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.For accurate identification, Why identification was not confirmed at molecular level e.g.16S rRNA and gyrB gene sequencing.16SrRNAgenesequencing is preferential over MALDI-TOF MS for definitive species identification.Clarify MALDI-TOF MS identification score in methodology and mention their limitations in discussion section.Scheduled identification and species characterization.Urine samples best to be cultivated on CLED agar media Lack of full Description of colony character and morphology of gram-stained film Update the references.Please rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Strongly supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes If this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?YesReviewer 2

Reviewer 1 recommendation and comments https
://doi.org/10.1099/acmi.0.000571.v1.4 © 2023 Alzubaidi L. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License.Dear Authors, Kindly find my comments below.1.Description of the case(s): Need more details for instance( Did the patient have a previous history of UTI)?, Did the infection appear before or after suprapubic?Do you think that the suprapubic had a role for causing infection?2.Presentation of results: Fig 1 for Antibiotics, ament title for inhibitory activity of antibiotic 3. Kindly mention to the Incubation temperaturePlease rate the quality of the presentation and structure of the manuscript GoodTo what extent are the conclusions supported by the data?Partially supportDo you have any concerns of possible image manipulation, plagiarism or any other unethical practices?NoIs there a potential financial or other conflict of interest between yourself and the author(s)?NoIf this manuscript involves human and/or animal work, have the subjects been treated in an ethical manner and the authors complied with the appropriate guidelines?Yes